使用复分析定性测量细菌生产和敏感性，以肺炎球菌细菌素
Summary
Competition in Streptococcus pneumoniae is mediated by bacteriocins, small antimicrobial peptides with inhibitory activity towards pneumococcus and other related species. Here we describe an optimized bacterial overlay assay that allows for the characterization of bacteriocin activity and inhibitory spectrum, bacteriocin-specific immunity, and detection of secreted quorum sensing peptides.
Abstract
Streptococcus pneumoniae colonizes the highly diverse polymicrobial community of the nasopharynx where it must compete with resident organisms. We have shown that bacterially produced antimicrobial peptides (bacteriocins) dictate the outcome of these competitive interactions. All fully-sequenced pneumococcal strains harbor a bacteriocin-like peptide (blp) locus. The blp locus encodes for a range of diverse bacteriocins and all of the highly conserved components needed for their regulation, processing, and secretion. The diversity of the bacteriocins found in the bacteriocin immunity region (BIR) of the locus is a major contributor of pneumococcal competition. Along with the bacteriocins, immunity genes are found in the BIR and are needed to protect the producer cell from the effects of its own bacteriocin. The overlay assay is a quick method for examining a large number of strains for competitive interactions mediated by bacteriocins. The overlay assay also allows for the characterization of bacteriocin-specific immunity, and detection of secreted quorum sensing peptides. The assay is performed by pre-inoculating an agar plate with a strain to be tested for bacteriocin production followed by application of a soft agar overlay containing a strain to be tested for bacteriocin sensitivity. A zone of clearance surrounding the stab indicates that the overlay strain is sensitive to the bacteriocins produced by the pre-inoculated strain. If no zone of clearance is observed, either the overlay strain is immune to the bacteriocins being produced or the pre-inoculated strain does not produce bacteriocins. To determine if the blp locus is functional in a given strain, the overlay assay can be adapted to evaluate for peptide pheromone secretion by the pre-inoculated strain. In this case, a series of four lacZ-reporter strains with different pheromone specificity are used in the overlay.
Introduction
肺炎链球菌 ，鼻咽部的多种微生物社会的共同殖民者，能够通过生产细菌产生抗菌肽（细菌素）与其他肺炎球菌和密切相关的物种竞争。每一个完全测序肺炎球菌菌株检测到包含最新版本的B acteriocin- 升 IKE p eptide轨迹，BLP的。细菌素生产由肺炎球菌已被证明是既在体外的结果，并在体内竞争1-4重要。竞争的动力学是由沿与同源免疫蛋白多样的细菌素的表达响应于一个特定的肽信息素的影响。感应的BLP轨迹是由一个群体感应系统，其中的肽信息素，BLPC，结合到传感器激酶，BlpH控制，并开始导致信号级联转录BLP轨迹5,6。有BLPC四个等位基因变异，每绑定到其同源BlpH 6。对于细胞生存其自身细菌素的影响，其编码同源免疫蛋白的基因被转录于同一操纵子作为各细菌的基因。如果竞争者应变是不能够上调BLP轨迹响应于外源信息素，或如菌株缺少所需的保护的特定免疫基因发生杀伤。该转运体复合物，BlpAB是所必需的肽信息素的分泌，加工和细菌的肽2,4。最近，它已被证明，肺炎球菌菌株的显著一部分是“骗子”，这意味着他们在blpA基因，使它们无法分泌信息素和细菌素1,2保守突变。这些菌株能够外感BLPC 2所分泌的嘶鸣回应无聊株允许生产的免疫蛋白。
虽然细菌素，从上清液粗制剂可以从生产者菌株用生化方法步骤，以达到纯度来制备，这种方法是不实用的筛选分离的大的集合，如果细菌素的表达水平是低的肉汤生长培养2,7- 9。覆盖测定法作为一种快速的方法来调查可能存在的在体外的菌株之间的竞争动态。要做到这一点，肺炎球菌菌株是预先接种到琼脂平板，让它生长，以达到局部细菌浓度足以诱导BLP轨迹。第二应变，然后接种到熔融的软琼脂溶液中，然后施加在预接种菌株来测试灵敏度。以评估为BLP轨迹（独立抑制活性）的活性，菌株可与一系列日来覆盖稀土前面所述BLPC记者株。这些菌株被构造成包含三个不同blpH等位基因是由肺炎链球菌分泌的三种最常见的BLPC信息素响应之一。记者菌株具有一个集成的lacZ基因，该基因是一个BlpH依赖的启动子的控制下，并携带的缺失在BLPC基因，防止自励10,11。
Protocol
Representative Results
Discussion
这个覆盖测定法是一种快速的方式来确定用于细菌素生产活动的范围，并表明肺炎球菌菌株之间可能出现的竞争性相互作用。覆盖测定法可适于用于筛选多株为细菌素生产上的单个板。我们已通过使用一个48针复制器从一个半到96孔板的接种SBA板成功筛选大应变的集合与该测定。隔夜培养后，成长正在使用的复制和穿孔板与复制的引脚的琼脂表面转移到TSA的板块。
被调节在像?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work has been supported by Elizabeth E. Kennedy Research Award.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Trypticase Soy Agar with 5% Sheep Blood | Fisher | B21261X | For growth from freezer cultures |
| Catalase | Sigma | C100-500mg | 4,741 Units per plate |
| Todd Hewitt Broth + Yeast Extract (THY) | Fisher | DF0492-17-6 | 30 g of THB, 5 g yeast extract, 1 L ddH20 autoclave |
| Trypticase Soy Broth + Agar plates (TSA) | Fisher | B11768 | 30 g of TS, 15 g agar, 1 L ddH20 autoclave |
| 5-Bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) | Sigma | B4252-50MG | Dissolve in dimethylformamide |
| Petri dishes | Fisher | FB0875712 | |
| Spectrophometer | Measures the OD620 of cultures | ||
| 37 °C water bath | |||
| 37 °C incubator with 5% CO2 | |||
| 15 ml conical tube |
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